Changes in the Pharmacokinetics of Ciprofloxacin and Fecal Flora During Administration of a 7-Day Course to Human Volunteers

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1984, P /84/ $02.00/0 Copyright X3 1984, American Society for Microbiology Vol. 26, No. 5 Changes in the Pharmacokinetics of Ciprofloxacin and Fecal Flora During Administration of a 7-Day Course to Human Volunteers W. BRUMFITT,* ITALIA FRANKLIN, DEIDRE GRADY, J. M. T. HAMILTON-MILLER, AND A. ILIFFE Department of Medical Microbiology, The Royal Free Hospital School of Medicine, Hampstead, London NW3 2QG, England Received 26 May 1984/Accepted 27 August 1984 Twelve male subjects, aged 19 to 40 years, shown to be healthy by examination and laboratory tests, took 500 mg of ciprofloxacin every 12 h for 7 days. After the first and the last dose, blood and urine samples were taken and drug concentrations were determined by bioassay. There was a significant buildup in mean concentrations in serum from day 1 to day 7; mean peak levels (attained after 1 to 2 h) were 1.9 and 2.8,ug/ml, respectively. The terminal half-life was 3.5 to 4 h. About 40% of the drug was excreted into the urine during the 12-h period after dosing; minimum mean concentrations in urine were 105,ug/ml on day 1 and 174,ug/ml on day 7. Considerable amounts of ciprofloxacin were found in the feces on day 7 (185 to 2,220,Lg/g). Marked changes in the aerobic part of the fecal flora were observed as a result of taking ciprofloxacin: coliforms were absent on day 7, and concentrations of streptococci and staphylococci were significantly reduced. There was no overgrowth by yeasts. One week later the fecal flora had returned to a state similar to that found before treatment. Anaerobes were little affected quantitatively but acquired resistance to ciprofloxacin. Side effects were mild and transient. Ciprofloxacin is a new synthetic broad-spectrum antimicrobial agent belonging to the 4-quinolone group and more active in vitro than norfloxacin (7, 12). Preliminary pharmacokinetic studies suggest a peak level in serum of ca. 2 [ig/ml 1 h after an oral dose of 500 mg, a half-life of ca. 3 h, and a urinary recovery every 12 h of ca. 30% (6). In the present study we have investigated the pharmacokinetic behavior and metabolism of ciprofloxacin in volunteers after a full therapeutic course (500 mg every 12 h for 7 days), and also its effect on the fecal flora. MATERIALS AND METHODS Volunteers. The purpose of the study was explained to 13 apparently healthy male subjects, none of whom worked in a hospital or had entered a hospital for 6 months before or during the study. Informed consent was obtained in writing. A full medical history and a detailed physical medical examination was carried out on each volunteer before the study started, and samples of blood and urine were analyzed to ensure that biochemical and hematological parameters fell within the normal ranges (see below for details). One volunteer (no. 4) found to suffer from insulin-dependent diabetes was excluded. Thus the study involved 12 healthy male subjects, whose ages ranged from 19 to 40 years (mean, 24.8), weighing between 57.3 and 95.5 kg (mean, 73.3). Pharmacokinetic profiles. Subjects fasted for at least 8 h before taking the first tablet of ciprofloxacin at 0800 h. The bladder was emptied, but a sample was kept for a base-line analysis. Blood was taken just before swallowing a 500-mg tablet of ciprofloxacin and at the following intervals thereafter: 30 min, and 1, 2, 3, 6, and 12 h. Each subject was closely observed during the first 30 min, including taking the blood pressure, pulse, and respiration rate at 15 and 30 min. If subjects did not complain spontaneously during the first 30 min, they were asked whether they had experienced any discomfort or other untoward event. The bladder was emp- * Corresponding author. 757 tied after 3, 6, and 12 h; all urine passed was collected; and the volume was measured. At 12 h the volunteers took another 500-mg tablet of ciprofloxacin; this dose was repeated at 12-h intervals. At 0800 h on day 7 (subjects having fasted for at least 8 h), another pharmacokinetic profile was carried out immediately after the subjects had taken the 500- mg tablet. Changes in fecal flora. Volunteers provided specimens of feces on the day before the first tablets were taken, on the last day of the treatment course, and 1 week later (day 14 of the study). Quantitative bacteriological estimations of the predominant fecal flora were made as described below, and the ciprofloxacin concentrations were assayed. Storage of specimens. Urine samples were mixed thoroughly, volumes were recorded, and two separate 20-ml samples were kept. Blood samples were allowed to clot, and serum was separated by centrifugation after the clot had retracted. The serum was divided into two equal parts, and both were saved. All the above specimens were stored at -20 C until used. One duplicate serum sample was used for bioassay, and the other was used for high-pressure liquid chromatographic assay. Volunteers were asked to send specimens of feces at least the size of a large pea to the laboratory. Fecal specimens were processed immediately on arrival in the laboratory. It was found that if the specimen had been left undisturbed before analysis no change in the major components of the flora occurred in a 24-h time lapse. This is in agreement with the findings of Crowther (5). Laboratory safety studies. The following tests were carried out before starting the study and on day 7 (viz., the day on which the course of tablets was completed): full blood count including indices, total and differential leukocyte count, platelet count, and microscopic examination of the blood film. Biochemical parameters included estimation of the fasting blood glucose, aspartate transaminase, alkaline phosphatase, y-glutamyltransferase, total bilirubin, total protein,

2 758 BRUMFITT ET AL. serum albumin, serum globulin, K+, Na+, Cl-, bicarbonate, urea, and creatinine. Urine was examined microscopically and tested for protein, glucose, ph, ketones, and blood with Labstix (Ames Div., Miles Laboratories). At the end of the study volunteers were also questioned in detail about any adverse effects experienced during the 7- day course of tablets. Microbiological assay. Antimicrobial activity was estimated by a hole-plate method. Klebsiella pneumoniae ATCC was grown overnight, and 10 ml of a 1:104 dilution was spread on the surface of Iso-Sensitest agar (Oxoid Ltd.) contained in large assay plates (24 by 24 cm; Nunc). Excess fluid was removed, and plates were dried for 30 min before holes were cut. The standard, ciprofloxacin hydrochloride monohydrate (1 g= 855 mg free base; supplied by Bayer UK Ltd.), was dissolved in phosphate-buffered saline (ph 7.3; Dulbecco 'A'; Oxoid BR14a), and concentrations of 2, 1, 0.5, 0.1, and 0.03,ug/ml were put onto each plate, in ANTIMICROB. AGENTS CHEMOTHER. duplicate. Serum and urine samples were diluted as required in phosphate-buffered saline and plated out in duplicate. Preliminary experiments showed that zone sizes were the same whether pooled serum or phosphate-buffered saline was used as the diluent. Plates were read after overnight incubation at 37 C. Analysis of pharmacokinetic data. Individual profiles were plotted on both arithmetic and logarithmic scales. The former was used to determine the area under the curve between 0 and 12 h (AUCo12) by cutting and weighing. The terminal serum half-life (t1/2) was determined for each volunteer from the logarithmic plots, and the P phase (, = 0.693/tl/2) elimination constant was calculated. AUCO.. was evaluated from the formula AUCO0 = AUC.12 + C,tI where C, equals the serum concentration at 12 h. Urinary excretion was calculated from the concentration of drug in the urine and the urine volume. Statistical significance was estimated by the Student t test. TABLE 1. Concentrations of ciprofloxacin (jig/ml) in serum' p.g of ciprofloxacin per ml of serum at following hours after dosage Volunteer no. Day Mean SD P <(0.05 <0.01 <0.05 NSb <0.02 <0.005 <0.05 (days 1 vs 7) Twelve volunteers were given 500 mg of ciprofloxacin orally every 12 h for 7 days. b NS, Not significant.

3 VOL. 26, 1984 PHARMACOKINETICS OF CIPROFLOXACIN FECAL FLORA 759 TABLE 2. Pharnmacokinetic parameters for serum concentrations Mean (SD) Day Half-life Time to ALJC (j±g * h/mi) (h) peak (h) 0-12 h (0.56) 1.45 (0.91) 10.7 (3.5) 11.8 (3.6) 7 4 (0.31) 1.8 (0.47) 16.6 (4.6) 18.8 (5.8) p <0.01 NSa <0.005 <0.005 a NS, Not significant. Fecal flora analysis. A weighed portion of feces (ca. 200 mg) was created with a Colworth Stomacher (supplied by A. J. Seward) in 9 volumes of 0.05% yeast extract made up inmm dithiothreitol, and decimal dilutions were made in the same fluid up to Volumes (0.1 ml) of appropriate dilutions (2) were spread over the whole surface of plates of the following media: MacConkey no. 3 (for coliforms), 6.5% NaCi-esculin (for staphylococci), nalidixic acid (200 plg/ml) blood agar (for streptococci), Anaerobic Blood Agar Base (GIBCO Europe) supplemented with 5% whole horse blood and 250,ug neomycin per ml (for anaerobes), and molybdatesucrose agar (for yeasts). Colonies which grew were counted and identified as far as possible, with API 20E for ferrhentative gram-negative bacilli, Unitek N/F for nonfermentative bacilli, An-Ident disks (Oxoid) for anaerobes, conventional methods for gram-positive species (4), and Uni-Yeast-Tek for yeasts. The methods used meant that the lower limit of detection was 100 organisms per g of feces. Statistical analysis was by the t test carried out on logarithms of viable counts. Sensitivity testing was carried out on Iso-Sensitest agar plus 10% lysed horse blood. Tests were carried out against four substances: nalidixic acid (30,.g), cinoxacin (30,ug), norfloxacin (10,ug), and ciprofloxacin (5,ug). Resistance was recorded when there was no zone, and sensitivity was recorded when the zone exceeded 12 mm; the vast majority of readings were definitive. TABLE 3. Concentrations of ciprofloxacin in urine and percentage of dose excreted and fecal concentrations at various time intervalsa,ug of ciprofloxacin per ml of urine (cumulative % of dose) at,ug of ciprofloxacin Volunteer Dosage following h after dosage per g of feces no. day immediately posttreatment (20.4) 60 (25.2) 80 (34.8) 1, (36) 390 (59.4) 190 (78.4) (8.3) 250 (18.3) 140 (28.1) (6.3) 152 (8.7) 100 (14.3) (15.2) 12 (18.1) 25 (22.8) 1, (15) 25 (21) 70 (28) (4.7) 95 (6.4) 170 (12.5) 2, (9.2) 400 (22) 300 (30.4) (40.8) 740 (55.6) 240 (66.2) (27.8) 150 (31.4) 110 (39.3) (16) 10 (18.3) 10 (20.6) (6.8) 10 (9.7) 13 (10.4) (17.6) 75 (35.6) 270 (78.8) 2, (20) 25 (26.5) 100 (41.5) (14.5)b 80 (19.6) 1, (25) 120 (28.6) 80 (34) (7.4) 20 (7.9) 61 (11.5) (5.9) 550 (23.5) 115 (35.5) (27) 120 (33) 70 (40.7) 1, (24) 250 (39) 100 (51) (7.6) 59 (12.3) 27 (17.9) 1, (7.2) 300 (31.2) 300 (49.2) (14.5) 200 (24.5) 120 (40.1) ,200 (24) 600 (42) 150 (57) Mean (19.2) 162 (26.2) 105 (38.6) (22.6) 291 (33.6) 174 (46) SD (12.3) 253 (16.2) 85 (24.8) (12.4) 239 (16.4) 100 (21.8) a TWelve volunteers were given 500 mg of ciprofloxacin orally every 12 h for 7 days. b Specimens pooled in error; thus, this value represents the 0- to 6-h specimen.

4 760 BRUMFITT -ET AL. TABLE 4. Effect of ciprofloxacin on fecal floraa Day Flora Value Pretreatment 7 14 Coliforms Mean 7.24 < SD Range <2 < Fecal streptococci Mean b 6.93 SD Range < Other streptococcic Mean b 4.22 SD Range <2-6.7 < < Staphylococci Mean b 3.07b SD Range < <2-2.7 < Yeasts Mean SD Range <2-5 < <2-4.6 Anaerobesd Mean SD Range a Means, standard deviations, and ranges of log1o counts per gram of feces of various microbial species pretreatment and on days 7 and 14. b Count was significantly lower than that at pretreatment (P < 0.05). c Usually Lancefield group C. d Microaerophilic organisms did not grow in the presence of 250 p.g of neomycin per ml. Differences in incidence of resistant strains were analyzed by the x2 test, with Yates correction applied if necessary. RESULTS Serum levels. The serum levels observed at various times in each of the 12 volunteers on days 1 and 7 of treatment are shown in Table 1. The pharmacokinetic parameters derived from these data are shown in Table 2. It can be seen that there was a buildup during the treatment period, as on day 7 concentrations were significantly higher at all sampling times (except 3 h) than they were on day 1. As a result, the area under the curve was greater on day 7 (Table 2). An increase in half-life was also observed. Urine excretion. High mean concentrations of ciprofloxacin were found in the urine (Table 3), and the mean trough level was in excess of 100,ug/ml (cf. MIC for Escherichia coli [0.1,ug/ml]). Although both the concentrations and the percentage of the given dose of ciprofloxacin excreted in 12 h were higher on day 7 than on day 1, differences were not significant. This was because of the large standard deviation, which resulted both from biological variation and from the greatly differing amounts of urine passed by the volunteers. High-pressure liquid chromatography. High-pressure liquid chromatography was carried out by D. Beermann at Bayer Laboratories (Wuppertal, Federal Republic of Germany), and his findings will be reported in detail elsewhere. Particular attention was paid to looking for metabolites in both serum and urine, but none was found. Concentration in feces. No antibacterial activity was found in any specimen obtained pretreatment or on day 14 (1 week posttreatment). However, high concentrations were present in the fecal specimens passed on the last day of treatment. Individual values ranged from 185 to 2,220 p.g/g, with a mean value of 891 (standard deviation, 624). It thus is clear that a ANTIMICROB. AGENTS CHEMOTHER. substantial amount of ciprofloxacin is present in the gut after oral administration. Effect on fecal flora. The 7-day course of ciprofloxacin caused a marked alteration on the composition of the fecal flora. Changes in the main groups of organisms studied are shown in Table 4. The most important finding was that coliforms had been entirely abolished from the feces by day 7; however, they had returned by day 14 in all but one subject. Pretreatment, the predominant coliform was Escherichia coli in 10 subjects and Enterobacter cloacae and Klebsiella oxytoca in 1 subject each. On day 14, Escherichia coli predominated in six subjects, Citrobacter freundii predominated in three, and Enterobacter cloacae predominated in two. Both staphylococci and streptococci were significantly reduced in number at day 7, but again these had returned in almost all cases by day 14. Numbers of obligate anaerobes and of yeasts did not alter significantly as a result of ciprofloxacin treatment. The fecal flora at day 7 (the final day of treatment) consisted of streptococci and anaerobes in four subjects, streptococci and yeasts in two, yeasts only in two, staphylococci, streptococci, and yeasts in two, anaerobes only in one, and yeasts and anaerobes in one. A detailed analysis of the fecal flora is available on request from the authors. All coliforms that recolonized the gut after the termination of treatment were like the original strains isolated, sensitive to ciprofloxacin (Table 5), as well as to norfloxacin, cinoxacin, and nalidixic acid. All staphylococci and streptococci originally present as gut flora were sensitive to ciprofloxacin, and the incidence of resistance was not significantly higher in those strains isolated on days 7 and 14 (as expected, all gram-positive strains were resistant to cinoxacin and nalidixic acid). However, the anaerobic strains changed from being mainly sensitive (75%) to ciprofloxacin pretreatment to being mainly resistant (only 12% were sensitive) after treatment (Table 5); this change was statistically significant (P < 0.001). It was of interest that most of the anaerobes were originally resistant to norfloxacin. Adverse effects. No subject complained of any adverse effect immediately after taking the tablets. Four subjects reported that they had experienced excessive intestinal flatus with an unpleasant odor starting at day 2 of treatment, but this disappeared before day 7. There were no significant changes in any of the measured biochemical or hematological parameters, and the results of urine analysis on day 7 remained normal. DISCUSSION Despite the great interest generated by the several new agents belonging to the 4-quinolone family, reflected by TABLE 5. Incidence of resistance to ciprofloxacin in various moieties of fecal flora during and after a 1-week course of ciprofloxacin No. of strains resistant to ciprofloxacin/total no. of strains isolated Bacterial type Day Pretreatment 7 14 Coliformsa 0/19 None isolated 0/24 Staphylococci 0/13 2/4 1/7 Streptococci 0/29 3/13 0/33 Anaerobes 9/36 26/30 23/26 a Comprising Escherichia coli, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, and Citrobacter freundii.

5 VOL. 26, 1984 PHARMACOKINETICS OF CIPROFLOXACIN FECAL FLORA 761 many publications of their in vitro properties, there have been very few papers on the pharmacokinetics of these compounds in humans. Adhami et al. (1) and Crump et al. (6) reported on the disposition of norfloxacin and ciprofloxacin, and Montay et al. (9) studied the pharmacokinetics of pefloxacin. However, all three studies were performed after only a single dose. Thus our study on a 4-quinolone seems to be the first involving a full therapeutic course. Our pharmacokinetic results show that ciprofloxacin behaves like norfloxacin (1) and pefloxacin (9). However, the half-life of the latter compound (9) is approximately double that of ciprofloxacin. Although ciprofloxacin is only moderately well absorbed when given by mouth (the mean urinary recovery was less than 40%), at the steady state a serum level of >1,ug/ml was maintained for 8 h. This is sufficient to inhibit the majority of gram-positive organisms and all gram-negative pathogens except some pseudomonads (6, 12), although not Bacteroides fragilis (12). In addition, ciprofloxacin has a very large volume of distribution (ca. 2 liters/kg [D. Beermann, personal communication]), which suggests that it may be concentrated in the tissues. Concentrations in urine were in the order of 100 to 200 times higher than levels in serum, and even allowing for the fact that ciprofloxacin is of reduced activity in urine (10), it is clear that the concentrations attained will be sufficient to inhibit all pathogens. Thus the oral dose of ciprofloxacin used here (500 mg every 12 h) appears to be more than adequate for therapeutic purposes. Even accounting for the relatively poor absorption of ciprofloxacin, levels in serum are low; amnpicillin, which is also 30 to 40% absorbed by mouth, gives peak levels of ca. 4,ug/ml after a 500-mg dose (3). One explanation for this is that ciprofloxacin has a larger volume of distribution than ampicillin (see above). Resolution of this point will have to await pharmacokinetic investigation after parenteral administration. We were particularly impressed by the effect of ciprofloxacin on the composition of the fecal flora. The total elimination of coliforms during the therapeutic course suggests that ciprofloxacin may be suitable for the prophylaxis of urinary infections. Further, the stability of the anaerobic moiety of the fecal flora is encouraging if one subscribes to the hypothesis of van der Waaij (11) that this is important in maintaining "colonization resistance." Thus, ciprofloxacin might be considered for prophylaxis in leukemic and other immunocompromised patients. Its success in these roles will depend upon the readiness with which common bacteria can acquire resistance, which will only become clear when the drug has had wider clinical use. The high concentrations of ciprofloxacin found in the feces were probably the result of poor absorption from the gastrointestinal tract rather than of recycling via the bile, as biliary excretion, although clinically significant, does not seem to be sufficient to account for the amounts of drug which can be recovered from the feces (unpublished data). There has been relatively little work published on fecal concentrations of antibiotics (see the review by George et al. [8]). However, it seems to be unusual for an antibiotic which is used for oral administration to appear (as ciprofloxacin does) in the feces in concentrations in excess of 1 mg/g. However, there is evidence for such findings for a few other antibiotics, such as tetracycline, erythromycin, and lincomycin (8). Although it is obvious that detailed clinical trials will be necessary to establish what part ciprofloxacin will play in clinical practice, our studies reported here suggest that 500 mg every 12 h will be a satisfactory dosage regimen for many purposes, and the effects we have observed on the fecal flora make a prophylactic role for this antibiotic worth pursuing if long-term toxicity tests prove to be satisfactory. Based on our experience with other antibiotics used for this purpose, it may be found that a smaller dose will prove to be more effective for prophylactic therapy. ACKNOWLEDGMENTS We are grateful to A. Westwood, I. Harding, D. Beerman, and A. Dahlhoff of Bayer UK and Bayer AG for a supply of ciprofloxacin tablets and many helpful discussions. LITERATURE CITED 1. Adhami, Z. N., R. Wise, D. Weston, and B. Crump The pharmacokinetics and tissue penetration of norfloxacin. J. Antimicrob. Chemother. 13: Brumfitt, W., and J. M. T. Hamilton-Miller Rifaprim (rifampicin plus trimethoprim): pharmacokinetics and effects on the normal flora of man. Biopharmaceut. Drug Disp. 2: Brumfitt, W., A. Percival, and M. J. Carter Treatment of urinary infections with ampicillin: a clinical trial. Lancet i: Cowan, S. T Cowan and Steel's manual for the identification of medical bacteria, 2nd ed. Cambridge University Press, Cambridge. 5. Crowther, J. S Transport and storage of faeces for bacteriological examination. J. Appl. Bacteriol. 34: Crump, B., R. Wise, and J. Dent Pharmacokinetics and tissue penetration of ciprofloxacin. Antimicrob. Agents Chemother. 24: Fass, R. J In vitro activity of ciprofloxacin (Bay o 9867). Antimicrob. Agents Chemother. 24: George, W. L., V. L. Sutter, and S. M. Finegold Toxigenicity and antimicrobial susceptability of Clostridium difficile, a cause of antimicrobial agent-associated colitis. Curr. Microbiol. 1: Montay, G., Y. Goueffon, and F. Roquet Absorption, distribution, metabolic fate, and elimination of pefloxacin mesylate in mice, rats, dogs, monkeys, and humans. Antimicrob. Agents Chemother. 25: Reeves, D. S., M. J. Bywater, H. A. Holt, and L. 0. White In vitro studies with ciprofloxacin, a new 4-quinolone compound. J. Antimicrob. Chemother. 13: van der Wanl, D Colonization resistance of the digestive tract: clinical consequences and implications. J. Antimicrob. Chemother. 10: Wise, R., J. M. Andrews, and L. J. Edwards In vitro activity of Bay 09867, a new quinolone derivative, compared with those of other antimicrobial agents. Antimicrob. Agents Chemother. 23: